Ameliorative effect of a standardized polyherbal combination in methotrexate-induced nephrotoxicity in the rat

Figures & data

Figure 1. HPLC chromatogram of hydroalcoholic extract of (a) B. diffusa, (b) R. emodi, (c) C. nurvala, (d) N. nucifera, (e) optimized combination (ABCD) and (f) standards recorded at 254 nm.

Table 1. HPLC fingerprinting of hydroalcoholic extract and polyherbal combination.

Rt (min)	B. diffusa	R. emodi	N. nucifera	C. nurvala	Combination (ABCD)
1.942	+	+	+	+	+
2.746	−	−	+	+	+
2.769	+	+	−	−	+
3.075	+	+	+	+	+
3.428	+	+	+	+	+
4.184	+	+	+	+	+
4.34	−	+	−	−	+
5.153	+	+	−	−	+
5.192	−	−	+	+	+
5.303	+	+	+	+	+
5.39	+	−	−	+	+
5.421	−	−	+	−	+
5.451	−	+	+	+	+
5.601	+	−	+	+	+
5.618	−	+	+	−	+
5.676	+	+	−	+	+
5.869	+	+	+	+	+
5.986	+	+	+	+	+
6.06	−	−	+	−	+
6.12	−	−	+	+	+
6.212	+	+	+	+	+
6.271	−	−	+	+	+
Emodin (Rt 6.415)	−	+	−	−	+
6.523	+	+	+	+	+
6.684	−	−	+	−	−
6.755	+	+	+	−	+
6.874	−	+	+	+	+
7.026	+	+	+	+	+
7.14	−	+	+	−	+
7.254	−	−	+	+	+
7.316	+	+	−	+	+
7.427	−	−	+	−	+
7.498	+	+	+	−	+
7.642	−	−	+	+	+
7.726	+	+	+	+	+
7.869	−	−	+	−	−
7.985	+	−	−	−	+
8.073	+	+	+	+	+
8.214	+	+	−	+	+
8.305	+	+	+	−	+
8.407	+	−	−	+	+
8.639	−	+	+	+	+
8.822	+	+	−	+	+
8.936	+	−	+	−	+
9.062	−	−	+	+	+
9.15	+	+	+	+	+
9.484	+	+	+	+	+
9.593	+	−	−	+	+
9.776	−	+	+	+	+
9.896	−	+	+	−	+
9.94	+	+	+	+	+
10.196	+	+	+	−	+
10.262	+	−	−	−	+
10.354	+	+	+	+	+
Quercetin (Rt 10.51)	+	+	+	+	+
10.674	+	−	+	+	+
10.809	−	+	−	+	+
10.909	+	+	+	+	+
11.182	+	+	−	+	+
11.342	−	−	+	+	+
11.591	+	−	−	+	+
11.695	+	+	−	+	+
11.95	−	+	−	+	+
12.061	+	−	−	+	+
12.242	−	−	−	+	−
12.499	−	−	−	+	−
12.702	+	0.9	−	+	+
12.801	−	−	−	+	−
12.988	−	−	−	+	−
13.079	+	+	+	+	+
13.22	−	−	−	+	−
13.386	−	+	−	+	−
13.579	−	−	−	+	+
13.835	−	−	−	+	−
13.937	−	−	−	+	+
Boeravinone (Rt 14.14)	+	−	−	−	+
14.452	−	+	+	+	+
14.585	−	+	−	+	+
14.751	−	−	−	+	+
14.932	−	−	−	+	+
15.095	−	−	−	+	−
15.422	−	−	−	+	+
15.696	−	−	−	+	−
15.993	−	−	−	+	+
16.175	−	−	−	+	−
16.558	+	+	+	+	+
16.678	−	−	−	+	+
16.856	−	−	−	+	+
17.01	−	−	−	+	−
17.229	−	−	−	+	−
17.454	−	−	−	+	+
17.803	−	−	−	+	+
18.004	−	−	−	+	+
18.411	−	−	−	+	+
18.49	−	−	−	+	+
18.939	−	−	−	+	+
19.362	+	+	+	+	+
19.724	−	+	+	+	+
19.881	−	+	−	+	+
20.138	−	−	−	+	+
20.335	−	+	−	+	+
20.629	−	−	−	+	−
20.758	−	−	−	+	+
20.996	−	−	−	+	+
21.302	−	−	−	+	−
21.458	−	−	−	+	+
21.703	−	−	−	+	+
22.107	−	−	−	+	+
22.253	−	−	−	+	−
22.899	−	−	−	+	−
23.373	−	−	−	+	+
23.707	+	+	+	+	+
Lupeol (Rt 23.85)	−	−	−	+	+
24.376	−	−	−	+	+
24.823	−	+	+	+	+
25.034	−	−	−	+	−
25.373	−	−	−	+	−
25.611	−	−	−	+	+
26.159	−	−	−	+	+
28.751	+	+	+	+	−
29.195	+	−	−	−	+
Total (122)	47	53	52	104	105

Figure 2. Phenolic and flavonoid content hydroalcoholic extract of different plants.

Table 2. The predicted and experimental value for % yield of extraction trials carried out by Box-Behnken’s response surface design for selected plant materials.^a

Run	A	B	C	N. nucifera		B. diffusa		C. nervala		R. emodi
				Actual	Predicted	Actual	Predicted	Actual	Predicted	Actual	Predicted
1	6.00	12.50	70.00	19.8	18.34	19.8	18.34	13.64	13.99	27.01	25.69
2	7.00	12.50	62.50	15.24	15.50	15.24	15.50	14.00	14.08	30.25	30.02
3	6.00	5.00	70.00	11.66	11.73	11.66	11.73	15.20	15.12	21.50	23.51
4	7.00	8.75	70.00	11.84	13.03	11.84	13.03	10.76	10.41	16.80	19.90
5	5.00	5.00	62.50	8.54	8.28	8.54	8.28	15.12	15.34	15.60	18.58
6	6.00	8.75	62.50	10.65	10.69	10.65	10.69	13.89	14.38	18.90	20.79
7	7.00	5.00	62.50	11.56	10.29	11.56	10.29	18.80	18.31	31.10	30.62
8	6.00	8.75	62.50	10.7	10.69	10.7	10.69	14.88	14.66	30.70	29.13
9	5.00	8.75	70.00	8.98	9.17	8.98	9.17	14.80	14.23	24.60	23.95
10	6.00	8.75	62.50	10.68	10.69	10.68	10.69	14.39	14.25	19.40	15.43
11	6.00	5.00	55.00	8.95	10.41	8.95	10.41	17.01	17.15	28.95	31.77
12	6.00	8.75	62.50	10.7	10.69	10.7	10.69	14.00	14.57	28.50	27.99
13	6.00	12.50	55.00	12.6	12.53	12.6	12.53	14.40	14.42	25.60	24.78
14	5.00	8.75	55.00	7.8	6.61	7.8	6.61	14.40	14.42	25.60	24.78
15	6.00	8.75	62.50	10.7	10.69	10.7	10.69	14.40	14.42	25.60	24.78
16	5.00	12.50	62.50	10.54	11.81	10.54	11.81	14.40	14.42	25.60	24.78
17	7.00	8.75	55.00	8.65	8.46	8.65	8.46	14.50	14.42	25.60	24.78

A: extraction time (hour); B: drug solvent ratio (v/v); C: alcohol water ratio (v/v).

^aLevels for extraction time are 5 h (−1), 6 h (0) and 7 h (+1); for drug solvent ratio (%v/v) are 5 (%v/v) (−1), 8.75 (%v/v) (0) and 12.5 (%v/v) (+1); for alcohol water ratio (%v/v) are 55 (%v/v) (−1), 62.5 (%v/v) (0) and 70 (%v/v) (+1).

Table 3. The predicted and experimental value for antioxidant potential (% of DPPH free radical scavenged) of extraction trials carried out by Box-Behnken’s response surface design for selected plant materials.^a

Run	A	B	C	N. nucifera		B. diffusa		C. nervala		R. emodi
				Actual	Predicted	Actual	Predicted	Actual	Predicted	Actual	Predicted
1	6.00	12.50	70.00	80.65	76.50	80.65	76.50	68.67	71.48	84.99	84.63
2	7.00	12.50	62.50	48.65	50.97	48.65	50.97	77.98	78.57	81.58	81.14
3	6.00	5.00	70.00	70.65	69.48	70.65	69.48	74.89	73.30	82.58	83.02
4	7.00	8.75	70.00	68.98	70.81	68.98	70.81	76.58	72.77	82.69	83.05
5	5.00	5.00	62.50	47.56	45.24	47.56	45.24	66.02	63.66	77.56	77.30
6	6.00	8.75	62.50	65.46	65.50	65.46	65.50	77.89	77.75	68.90	68.72
7	7.00	5.00	62.50	51.26	50.60	51.26	50.60	81.65	81.12	78.89	79.07
8	6.00	8.75	62.50	65.39	65.50	65.39	65.50	71.89	73.58	83.92	84.19
9	5.00	8.75	70.00	71.65	75.14	71.65	75.14	78.56	77.96	75.58	76.20
10	6.00	8.75	62.50	65.45	65.50	65.45	65.50	59.65	63.45	65.99	65.81
11	6.00	5.00	55.00	51.26	55.41	51.26	55.41	74.52	72.09	74.11	74.29
12	6.00	8.75	62.50	65.65	65.50	65.65	65.50	80.65	82.62	85.60	84.98
13	6.00	12.50	55.00	55.65	56.82	55.65	56.82	75.90	74.03	79.80	79.64
14	5.00	8.75	55.00	54.26	52.43	54.26	52.43	75.90	74.03	78.90	79.64
15	6.00	8.75	62.50	65.56	65.50	65.56	65.50	72.50	74.03	79.89	79.64
16	5.00	12.50	62.50	52.65	53.31	52.65	53.31	72.60	74.03	79.80	79.64
17	7.00	8.75	55.00	63.25	59.76	63.25	59.76	72.60	74.03	79.80	79.64

A: extraction time (h); B: drug solvent ratio (v/v); C: alcohol water ratio (v/v).

^aLevels for extraction time are 5 h (−1), 6 h (0) and 7 h (+1); for drug solvent ratio (%v/v) are 5 (%v/v) (−1), 8.75 (%v/v) (0) and 12.5 (%v/v) (+1); for alcohol water ratio (%v/v) are 55 (%v/v) (−1), 62.5 (%v/v) (0) and 70 (%v/v) (+1).

Figure 3. Response surface plots showing relative effect of two extraction parameters for (a) B. diffusa (a_i: yield and a_ii: antioxidant activity); (b) R. emodi (b_i: yield and b_ii: antioxidant activity); (c) N. nucifera (c_i: yield and c_ii: antioxidant activity) and (d) C. nurvala (d_i: yield and d_ii: antioxidant activity); but other parameters were kept at constant levels.

Table 4. Statistical analysis of the models for antioxidant potential obtained from response surface methodology for extraction of selected plant.

Parameters	N. nucifera		B. diffusa		C. nervala		R. emodi
	Yield	Antioxidant	Yield	Antioxidant	Yield	Antioxidant	Yield	Antioxidant
Regression
Sum of square	119.33	1346.7	119.33	1346.7	39.75	406.01	308.36	445.7
Df	9	9	9	9	9	9	6	9
Mean squares	13.26	149.64	13.26	149.64	4.42	67.67	51.39	49.52
F value	8.83	13.13	8.83	13.13	20.01	9.39	8.8	145.59
p Value	0.0045	0.0013	0.0045	0.0013	0.0003	0.0012	0.0016	0.0001
Residual
Sum of square	10.51	79.8	10.51	79.8	1.54	72.06	58.37	2.38
Df	3	7	3	7	7	10	10	7
Mean square	3.50	11.40	3.50	11.40	0.22	7.21	5.84	0.34
Lack of fit test
Sum of square	10.51	79.76	10.51	79.76	1.54	58.72	58.37	1.69
Df	3	3	3	3	3	6	6	3
Mean squares	3.50	26.59	3.50	26.59	0.51	9.79	9.73	0.56
F value	73.00	25.25	73.00	25.25	256.1	2.93	24.56	3.29
CV %	0.62	0.55	0.62	0.55	3.21	3.63	9.75	0.74
R^2	0.919	0.944	–	–	0.96	0.849	0.84	0.99
Adequate precision value	10.99	5.50	–	–	21.92	11.12	10.539	42.84

Table 5. Optimized parameters and predicted dependent variables for extraction of selected plant materials.

	B. diffusa	R. emodi	N. lucifera	C. nurula
Extraction parameters
Extraction time (h)	5.05	7.00	6.43	5.35
Drug: Solvent (v/v)	11.99	6.32	10.68	11.79
Alcohol: Water (v/v)	58.24	67.97	68.99	68.99
Predicted value (Average ± SE mean)
% Yield	19.26 ± 1.79	30.73 ± 1.86	15.24 ± 0.75	16.37 ± 0.35
Antioxidant values	86.25 ± 1.22	81.59 ± 0.47	74.16 ± 2.84	83.46 ± 2.00
Experimental values at optimized parameters (Average ± SE mean)
% Yield	22.34 ± 2.84	29.78 ± 2.11	18.25 ± 1.45	17.25 ± 0.89
Antioxidant values	85.41 ± 4.56	81.65 ± 1.47	75.65 ± 1.35	81.56 ± 1.65

Figure 4. Screening of different combinations of extracts for (a) antioxidant activity and (b) XO inhibition potential.

Table 6. Effect of polyherbal combinations on kidney function parameters.^a

KFR parameters	Normal control	ABCD Sham	ABD Sham	Toxic control	ABCD 1	ABCD 2	ABD 1	ABD 2	Positive control
Urea (mg/dL)	35.6 ± 0.21	41.5 ± 1.21	42.5 ± 1.21	84.1 ± 1.21^###	52.2 ± 0.94**	41.5 ± 0.74***	59.5 ± 1.21*	54.3 ± 0.74**	69.1 ± 0.86*
Creatinine (mg/dL)	0.68 ± 0.11	0.75 ± 0.07	0.77 ± 0.05	1.59 ± 0.08^#	1.31 ± 0.08*	0.89 ± 0.11*	1.44 ± 0.08^ns	1.11 ± 0.01*	1.41 ± 0.02^ns
Uric acid (mg/dL)	2.67 ± 0.09	2.87 ± 0.08	2.68 ± 0.54	4.24 ± 0.05^##	3.26 ± 0.12*	2.24 ± 0.08*	3.19 ± 0.08^ns	2.89 ± 0.04*	2.98 ± 0.21*
Total protein (g/dL)	5.54 ± 0.07	5.61 ± 0.14	5.58 ± 0.65	7.59 ± 0.45^#	5.42 ± 0.11^ns	5.11 ± 0.05^ns	6.73 ± 0.45^ns	6.12 ± 0.05^ns	6.11 ± 0.31^ns
Albumin (g/dL)	2.89 ± 0.04	2.88 ± 0.11	2.79 ± 0.07	3.69 ± 0.04^#	3.24 ± 0.08^ns	2.75 ± 0.04*	3.64 ± 0.06^ns	2.89 ± 0.11*	2.88 ± 0.08*
Globulin (g/dL)	1.89 ± 0.06	1.94 ± 0.08	1.95 ± 0.04	3.72 ± 0.03^#	3.35 ± 0.06^ns	2.58 ± 0.06*	3.09 ± 0.05^ns	2.89 ± 0.05*	2.11 ± 0.09*
Total bilirubin (mg/dL)	0.28 ± 0.04	0.31 ± 0.05	0.32 ± 0.05	0.34 ± 0.02^ns	0.31 ± 0.04^ns	0.29 ± 0.04^ns	0.29 ± 0.02^ns	0.21 ± 0.03*	0.29 ± 0.05^ns
Direct Bilirubin (mg/dL)	0.17 ± 0.04	0.12 ± 0.02	0.15 ± 0.05	0.13 ± 0.02^ns	0.15 ± 0.05^ns	0.16 ± 0.07^ns	0.12 ± 0.01^ns	0.13 ± 0.04^ns	0.08 ± 0.07*
Indirect bilirubin (mg/dL)	0.11 ± 0.03	0.19 ± 0.02	0.17 ± 0.04	0.21 ± 0.03^ns	0.16 ± 0.04*	0.13 ± 0.05*	0.17 ± 0.06*	0.08 ± 0.04*	0.11 ± 0.04*

^aData were expressed as mean ± SEM. ^#Indicates data were compared with normal control. *Data were compared with toxic control. ***p < 0.001; **p < 0.01; *p < 0.1; ^nsp > 0.5; ^###p < 0.001; ^##p < 0.01; ^#p < 0.1.

Table 7. Effect of polyherbal combinations on oxidative markers in serum and in kidney homogenate.^a

Oxidative markers	Normal Control	ABCD Sham	ABD Sham	Toxic control	ABCD 1	ABCD 2	ABD 1	ABD 2	Positive control
Serum
SGOT (U/L)	35.4 ± 1.21	36.7 ± 1.31	34.6 ± 0.08	76.9 ± 0.08***	48.2 ± 0.08^##	39.4 ± 0.08^###	54.6 ± 0.08^#	42.5 ± 0.08^###	41.5 ± 0.08^###
SGPT (U/L)	21.2 ± 0.89	20.4 ± 0.78	21.9 ± 0.65	82.6 ± 1.24***	32.6 ± 0.12^###	28.6 ± 0.74^###	41.2 ± 0.15^###	35.4 ± 0.36^###	31.5 ± 0.61^###
Alkaline Phosphatase (U/L)	78.5 ± 0.45	79.2 ± 1.35	77.4 ± 1.25	212.5 ± 2.15***	125.6 ± 1.2^###	98.6 ± 0.74^###	135.4 ± 1.3^###	111.4 ± 1.1^###	97.6 ± 0.74^###
Catalase (µmol H2O2/L/min)	179.4 ± 1.5	176.5 ± 1.25	177.4 ± 1.5	255.6 ± 1.89***	198.6 ± 1.4^#	184.6 ± 1.4^###	201.4 ± 1.35^ns	194.5 ± 1.4^#	184.5 ± 1.2^###
Malonaldehyde (µmol/mL)	1.8 ± 0.045	1.8 ± 0.08	1.7 ± 0.04	4.11 ± 0.04***	2.4 ± 0.08^#	1.9 ± 0.05^##	2.7 ± 0.06^ns	2.1 ± 0.08^#	1.9 ± 0.08^##
Kidney homogenate
SGOT (U/L)	35.4 ± 0.78	36.7 ± 1.26	34.6 ± 0.15	76.9 ± 0.87***	48.2 ± 1.25^##	39.4 ± 0.46^###	54.6 ± 0.11^#	42.5 ± 0.16^##	41.5 ± 0.65^##
SGPT (U/L)	21.2 ± 0.44	20.4 ± 0.54	21.9 ± 0.48	82.6 ± 0.69***	32.6 ± 0.78^###	28.6 ± 0.25^###	41.2 ± 0.21^###	35.4 ± 0.65^###	31.5 ± 0.54^###
Alkaline Phosphatase (U/L)	78.5 ± 1.12	79.2 ± 1.12	77.4 ± 1.56	212.5 ± 1.45***	125.6 ± 0.9^##	98.6 ± 1.24^###	135.4 ± 1.23^##	111.4 ± 1.2^###	97.6 ± 1.36^###
Catalase (µmol H2O2/g tissue/min)	79.4 ± 1.45	78.5 ± 1.56	79.4 ± 0.89	135.6 ± 1.48**	94.5 ± 0.78^#	95.6 ± 0.98^#	105.6 ± 1.24^##	97.5 ± 0.68^#	88.6 ± 1.47^##
MDA (µmol/g wet tissue)	0.67 ± 0.03	0.68 ± 0.02	0.71 ± 0.03	2.8 ± 0.02*	1.4 ± 0.06^#	0.91 ± 0.04^#	1.78 ± 0.04^ns	1.1 ± 0.03	0.96 ± 0.03^#

^aData were expressed as mean ± SEM. *Data were compared with normal control. ^#Data were compared with toxic control. ***p < 0.001; **p < 0.01; *p < 0.1, ^nsp > 0.5; ^###p < 0.001; ^##p < 0.01; ^#p < 0.1.

Figure 5. Effect of combination on TNF-α of methotrexate-induced nephrotoxicity in rats. Data were expressed as mean ± SEM. *Data were compared with normal control. ^#Data were compared with toxic control. ***p < 0.001.

Figure 6. Microscopic images of kidney sections under light microscope (40×) after haematoxylin and eosin staining from animals of (a) normal control, (b) Sham control of combination ABCD, (c) Sham control of combination ABD, (d) methotrexate treated animals, (e) nephrotoxic rats treated with ABCD at a dose of 200 mg/kg, (f) nephrotoxic rats treated with ABCD rats at a dose of 300 mg/kg, (g) nephrotoxic rat treated with ABD combination at a dose of 250 mg/kg and (h) nephrotoxic rats treated with Neeri KFT tablet at a dose of 100 mg/kg.

Table 8. Effect of formulation histological parameters of methotrexate induced nephrotoxic rats.

Histological features	Normal control	ABCD Sham	ABD Sham	Toxic control	ABCD 1	ABCD 2	ABD 1	ABD 2	Positive control
Glomerular congestion	−	−	−	+++	+	−	−	−	−
Tubular casts	−	−	−	+++	+	−	+	+	−
Peritubular desquamation	−	−	−	+++	++	+	+	−	+
Epithelial desquamation	−	−	−	+++	+	−	++	+	+
Blood vessel congestion	−	−	−	+++	−	−	−	−	−
Interstitial oedema	−	−	−	+++	+	−	−	−	−
Inflammatory cells	−	−	−	+++	+	−	+	−	−

Figure 7. Three-dimensional interaction of XO proteins with (a) Boeravinone b, (b) emodin, (c) armepavine and (b) lupeol.